Topology design of 2D and 3D elastic material microarchitectures with crystal symmetries displaying isotropic properties close to their theoretical limits

This paper evaluates the effect that different imposed crystal symmetries have on the topology design of two-phase isotropic elastic composites ruled by the target of attaining extreme theoretical properties. Extreme properties are defined by the Cherkaev–Gibiansky bounds, for 2D cases, or the Hashin–Shtrikman bounds, for 3D cases. The topology design methodology used in this study is an inverse homogenization technique which is mathematically formulated as a topology optimization problem. The crystal symmetry is imposed on the material configuration within a predefined design domain, which is taken as the primitive cell of the underlying Bravais lattice of the crystal system studied in each case. The influence of imposing crystal symmetries to the microstructure topologies is evaluated by testing five plane groups of the hexagonal crystal system for 2D problems and four space groups of the cubic crystal systems for 3D problems. A discussion about the adequacy of the tested plane or space groups to attain elastic properties close to the theoretical bounds is presented. The extracted conclusions could be meaningful for more general classes of topology design problems in the thermal, phononic or photonic fields.Peer ReviewedPostprint (published version

A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state.Fil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Centeno, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Inhibition of the gastric H,K-ATPase by potassium competitive acid blockers

The gastric H,K-ATPase is a membrane protein found in the parietal cells of the stomach, where it couples H+ extrusion to the uptake of K+ , leading to the acidification of gastric juice (1). Acid-related diseases are an important public health issue where the mainstay of treatment has been the suppression of H,K-ATPase activity. As K+ plays a vital role in this catalytic cycle, for the dephosphorylation of the H,K-ATPase and the subsequent conformational changes, acid secretion can be inhibited by agents that are competitive with respect to K+ binding. This argument led in the past decades to the development of a new class of acid suppressants, known as potassium competitive acid blockers (P-CABs). Since a systematic investigation of enzyme-inhibition mechanisms has become a fruitful way to design and test new drugs, the effects of P-CABs-type inhibitors have been extensively studied analyzing how the apparent Michaelis and Menten parameters are affected (2). Working with the non-compartmentalized enzyme preparation, we analyzed the interactions between K+ , the H,K-ATPase, and two different inhibitors under steady state conditions. Our results from ATPase activity as a function of K+ concentration was described by a rational function where the maximal exponent on [K+] is 2. Data show that K+ , as a product, can inhibit the reaction steps that involve its release, which implies that ATPase activity would not obey the Michaelis-Menten equation. This can lead to mistakes when analysing the results according to variations in Vmax and KM . Here we propose a minimal model to describe the binding of K+ to different enzyme conformations and the inhibition by P-CABs compounds allowing a more realistic evaluation of their effects.Fil: Cerf, Nicole Talia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Valsecchi, Wanda M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaXLIX Reunión Anual de la Sociedad Argentina de BiofísicaArgentinaSociedad Argentina de Biofísic

Aluminum inhibits the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases by different mechanisms

Aluminum (Al 3+ ) is involved in the pathophysiology of neurodegenerative disorders. The mechanisms that have been proposed to explain the action of Al 3+ toxicity are linked to changes in the cellular calcium homeostasis, placing the transporting calcium pumps as potential targets. The aim of this work was to study the molecular inhibitory mechanism of Al 3+ on Ca 2+ -ATPases such as the plasma membrane and the sarcoplasmic reticulum calcium pumps (PMCA and SERCA, respectively). These P-ATPases transport Ca 2+ actively from the cytoplasm towards the extracellular medium and to the sarcoplasmic reticulum, respectively. For this purpose, we performed enzymatic measurements of the effect of Al 3+ on purified preparations of PMCA and SERCA. Our results show that Al 3+ is an irreversible inhibitor of PMCA and a slowly-reversible inhibitor of SERCA. The binding of Al 3+ is affected by Ca 2+ in SERCA, though not in PMCA. Al 3+ prevents the phosphorylation of SERCA and, conversely, the dephosphorylation of PMCA. The dephosphorylation time courses of the complex formed by PMCA and Al 3+ (EPAl) in the presence of ADP or ATP show that EPAl is composed mainly by the conformer E 2 P. This work shows for the first time a distinct mechanism of Al 3+ inhibition that involves different intermediates of the reaction cycle of these two Ca 2+ -ATPases.Fil: de Sautu, Marilina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Saffioti, Nicolas Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferreira Gomes, Mariela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Mangialavori, Irene Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Conformational changes produced by ATP binding to the plasma membrane calcium pump

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca2+ binding domain, we also studied the occlusion of Ca2+, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca2+ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.Fil: Mangialavori, Irene C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Ferreira Gomes, Mariela S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Saffioti, Nicolas A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentin

Regulation of plasma membrane calcium ATPase (PMCA) by actin cytoskeleton

The Plasma Membrane Calcium ATPase (PMCA) is a calmodulin-modulated P-type ATPaseresponsible for the maintenance of low intracellular concentrations of Ca2+ in mosteukaryotic cells. Our group have previously shown that purified actin can exert a dualmodulation on the activity of Ca2+-ATPase 4b isoform (hPMCA4b): F-actin inhibits it whileshort actin oligomers may contribute to its activation. These studies had to be performedwith purified proteins given the nature of the biophysical and biochemical approachesused.On the other hand, in HEK293 human cells that overexpressed PMCA2w/b isoform, theactin depolymerization upon Cytochalasin D (CytD) treatment significantly increasedPMCA2-mediated Ca2+ extrusion and when F-actin was stabilized using jasplakinolide,PMCA2w/b activity was completely abolished.In order to assess whether the functional interaction between the hPMCA4 isoform and theactin cytoskeleton may be of physiological relevance, we decided to further characterize itin the context of a living cell by monitoring in real-time the changes in the actinpolymerization and cytosolic Ca2+ concentration ([Ca2+]cyt). For this, hPMCA4 isoform wastransiently expressed in HEK293T cells. The dynamics of [Ca2+]cyt was performed usingthe fluorescent probe Fluo-4 and studying the alterations in [Ca2+]cyt generated by Ca2+release from the endoplasmic reticulum, and by extracellular Ca2+ entry through store-operated Ca2+ channels. The dynamics of actin polymerization was performed transientlyexpressing LifeAct-Ruby.Results show that the alteration of actin polymerization by CytD treatment significantlyincreased hPMCA4 activity (102%). On the other hand, in absent of CytD, actinpolymerization dynamics did not change after TG stimulus, while after Ca2+ stimulus, anactin reorganization was observed. This reorganization takes place at the same times thatthe hPMCA4 increases its activity, suggesting that hPMCA4 may be activated by actindepolymerization in the cells.Fil: Vigil, Maximiliano Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Picco, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Rinaldi, Debora Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rey, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferreira Gomes, Mariela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaXLIX Reunión Anual de la Sociedad Argentina de BiofísicaArgentinaSociedad Argentina de Biofísic

Testing divalent cations as essential activators of the ATPase activity and effect of ssRNA on the catalytic cycle of Zika Virus NS3 helicase

Zika Virus non structural protein 3 (NS3h) is a molecular motor that couples translocation along single stranded and unwinding of double-stranded RNA with the catalysis of the hydrolysis of nucleoside triphosphates (NTPs)[1]. ATPase activity of NS3h is dependent on the presence of magnesium, which is an essential activator. In this work we study the effect of single-stranded RNA on the ATPase activity of NS3h and investigated the ability of different divalent cations to replace the role of magnesium. ATP substrate curves were obtained at different concentrations of homopolyribonucleotide poly(A). ATPase activity of NS3h was enhanced by the presence of RNA, we propose and fit to the experimental data a kinetic model that describes such effect. In order to determine the ability of different divalent cations to replace magnesium as an essential activator on the catalysis of ATP hydrolysis by NS3h we performed ATPase activity measurements in media containing CaCl2, SrCl2, MnSO2 or FeSO4 in the absence of Mg2+. Activity was observed in the presence of both MnSO2 and CaCl2 and was non detectable in the case of SrCl2 and FeSO4.Fil: Mikkelsen, Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gebhard, Leopoldo German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Incicco, Juan Jeremías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Cababie, Leila Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Kaufman, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaXLIX Reunión Anual Sociedad Argentina de BiofísicaCiudad Autónoma de Buenos AiresArgentinaSociedad Argentina de Biofísic

E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na+ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na+-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+.Fil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Monti, José Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentin

Steady-state analysis of enzymes with non-Michaelis-Menten kinetics: The transport mechanism of Na + /K + -ATPase

Procedures to define kinetic mechanisms from catalytic activity measurements that obey the Michaelis-Menten equation are well-established. In contrast, analytical tools for enzymes displaying non-Michaelis-Menten kinetics are underdeveloped and transient-state measurements, when feasible, are therefore preferred in kinetic studies. Of note, transient-state determinations evaluate only partial reactions, and these might not participate in the reaction cycle. Here, we provide a general procedure to characterize kinetic mechanisms from steady-state determinations. We described non-Michaelis-Menten kinetics with equations containing parameters equivalent to kcat and KM and modeled the underlying mechanism by an approach similar to that used under Michaelis-Menten kinetics. The procedure enabled us to evaluate whether Na+/K+-ATPase uses the same sites to alternatively transportNa+ andK+. This ping-pong mechanism is supported by transient-state studies but contradicted to date by steady-state analyses claiming that the release of one cationic species as product requires the binding of the other (ternary-complex mechanism). To derive robust conclusions about Na+/K+- ATPase transport mechanism, we did not rely onATPase activity measurements alone. During the catalytic cycle, the transported cations become transitorily occluded (i.e. trapped within the enzyme). We employed radioactive isotopes to quantify occluded cations under steady-state conditions. We replaced K+ with Rb+ since 42K+ has a short half-life and previous studies showed that K+- and Rb+-occluded reaction intermediates are similar. We derived conclusions regarding the rate of Rb+-deocclusion that were verified by direct measurements. Our results validated the ping-pong mechanism and proved that Rb+-deocclusion is accelerated when Na+ binds to an allosteric, unspecific site, leading to a two-fold increase in ATPase activity.Fil: Monti, José Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentin

How to distinguish ligand-binding mechanisms: an example of conformational selection disguised as an induced fit

This report describes the implementation of a laboratory exercise for an advanced biochemistry or enzyme kinetics class at the undergraduate or graduate level, designed to improve understanding of protein conformational changes associated with the binding of a ligand. Students measure the fluorescence changes induced by the conformational transition of a glycoprotein (the Na,K-ATPase) upon addition of different ligands (Pi and BeF3 −) and analyse the results in order to determine the mechanism of the process. The results show that Pi and BeF3 − present opposite effects on the observed rate constants (kobs) with ligand concentration: kobs decreases with [Pi] and increases with [BeF3 −]. This observation, together with the frequently used assumption that binding occurs under rapid equilibrium, led to propose different models for ligand-induced conformational transitions: a conformational selection for Pi and an induced fit for BeF3 −. In this paper, we show that if the rapid-equilibrium approximation for ligand binding is not assumed, a conformational selection mechanism can account for the effects of both ligands. This active-learning exercise serves as the basis for discussing the consequences of not being extremely cautious when invoking approximations about not-very-well-known systems and the importance of a correct understanding of models assigned to chemical processes.Fil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Lopez Montes, Mónica Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin